Compensatory caspase activation in MPP+-induced cell death in dopaminergic neurons.

Many have hypothesized that cell death in Parkinson's disease is via apoptosis and, specifically, by the mitochondrial-mediated apoptotic pathway. We tested this hypothesis using a mouse dopaminergic cell line of mesencephalic origin, MN9D, challenged with the Parkinsonism-causing neurotoxin MPP+ (1-methyl-4-phenylpyridinium ion). Apoptosis was the main mode of cell death when the cells were subjected to MPP+ treatment under serum-free conditions for 24 h. Caspase-3 and caspase-9, however, were not activated, thus indicating the existence of alternate or compensatory cell death pathway(s) in dopaminergic neuronal cells. Using caspase inhibitors, we demonstrated that these pathways involve caspase-2, -8, -6 and -7. A time-course study indicated that activation of caspase-2 and -8 occurred upstream of caspase-6 and caspase-7. Upon MPP+ challenge, the apoptosis-inducing factor was translocated from the mitochondria into the MN9D cytosol and nucleus. These results suggest the existence of alternative apoptotic pathways in dopaminergic neurons.

The chaperonin containing TCP-1 (CCT) displays a single-ring mediated disassembly and reassembly cycle.

The chaperonin-containing TCP-1 (CCT) assists in the folding of actins and tubulins in eukaryotic cells. CCT is composed of 8 subunit species encoded by separate genes. CCT purifies as a single hetero-oligomeric protein complex of 950 kDa through multiple chromatographic and antibody affinity procedures. The CCT 16-mer contains 7 polypeptide species in equimolar amounts (CCTalpha, beta, gamma, delta, epsilon, zeta, eta), together with another subunit (CCTtheta) which is around half-molar. Here we show, by in vitro translation of CCT subunit mRNAs in rabbit reticulocyte lysate, that none of the CCT subunit proteins are themselves folded by CCT. However, the newly translated CCT subunits can incorporate into the endogenous CCT complex present in the lysate via a mechanism involving a nucleotide-dependent disassembly reaction to produce single-rings and then a reassembly reaction whereby free CCT subunits assemble onto these single-rings. This cycling behaviour is an inherent property of the CCT chaperonin complex and provides a powerful method for introducing single amino acid residue changes into this 8578 residue protein complex.

Elucidation of the subunit orientation in CCT (chaperonin containing TCP1) from the subunit composition of CCT micro-complexes.

A collection of chaperonin containing TCP1 (CCT) micro-complexes that are comprised of subsets of the constitutively expressed CCT subunits have been identified. These CCT micro-complexes have mol. wts ranging from 120 to 250 kDa and are present in cells at lower abundance (<5%) as compared with intact CCT. Biochemical characterization of these microcomplexes has shown that several are comprised of two different types of CCT subunit. Furthermore, it was observed that each subunit associates with only one or two other different types of subunit, suggesting that each subunit has fixed partners. This observation, together with CCT gene counting being concordant with the 8-fold structural symmetry, is consistent with predictions derived from analysis of the primary structures of these subunits concerning inter-subunit interactions, and implies a unique topology of the subunits constituting the torodial ring in CCT. The series of subunit-subunit association patterns determined from CCT micro-complexes has provided information to infer, from the 5040 (7!factorial) combinatorial possibilities, one probable subunit orientation within the torodial ring.